The objective of the proposed research is to define the clonal population structure and genetic relationships of pathogenic forms of Escherichia coli, with special reference to cytotoxin-producing strains that only recently have been linked to enteric disease in humans and animals. Although the mechanisms by which these bacteria cause disease is not fully understood, the ability to elaborate potent cytotoxins -- called Shiga-like toxins (SLTs) because of their functional similarity to Shiga toxin of Shigella dysenteriae - appears to play an important role in pathogenesis. One cytotoxigenic strain, serotype 0157:H7, is a newly recognized pathogenic clone that has recently spread through North America and caused severe outbreaks of hemorrhagic colitis and hundreds of sporadic cases of gastrointestinal illness. Because SLT-genes occur in distinctly related lineages of the E. coli population, they are ideal organisms for investigating the epidemiology, population genetics, and evolution of virulence factors in pathogenic forms. The proposed program extends ongoing research on the clonal nature of populations of cytotoxigenic E. coli, in the course of which we have discovered a close generic relationship between the 0157:H7 clone and a classical EPEC serotype (055:H7) that has long been associated with outbreaks of infantile diarrhea. This finding, as well as the observation that SLT genes occur in a diversity of E. coli genotypes, has motivated specific projects with the following aims: (1) to identify and characterize the major cytotoxigenic clones that account for a majority of cases of enteric disease; (2) to determine the generic relationships among the major cytotoxin-producing clone families and other pathogenic strains with similar virulence properties; (3) to investigate the extent of sequence divergence and horizontal transfer of Shiga-like toxin (SLT) genes in genetically distinct host strains; and (4) to explore the subclonal structure among isolates of the 0157:H7 clone using rapidly evolving insertion sequences. To address these aims, we will use multilocus enzyme electrophoresis to detect generic (allelic) variation in 20 enzyme-encoding chromosomal genes and, thereby, characterize the chromosomal genotypes of bacterial isolates. The system of chromosomal markers provides a genetic basis for assessing clonal relationships between pathogenic strains, determining the distribution of plasmid-home virulence factors across host strains, and delineating the major evolutionary branches within these phenotypic groups. The inferred genetic relationships among clonal genotypes will, in turn, provide a framework for the study of the divergence of SLT alleles based on the analysis of restriction fragment length polymorphisms and DNA sequences. The results will be used to test hypotheses regarding the clonal nature and origin of cytotoxigenic strains, and the significance of the horizontal transfer of SLT genes in nature. The long-term objective is to establish a genetic classification of strains that represent the major pathogenic lineages of E. coli associated with human and animal infections.